Tuberculosis diagnostic test

ABSTRACT

A method of diagnosing in a host infection by or exposure to a mycobacterium which expresses ESAT-6 comprising (i) contacting a population of T cells from the host with one or more peptides or analogues selected from the peptides represented by SEQ ID NO:1 to 11 and analogues thereof which can bind a T cell receptor which recognises any of the said peptides, and (ii) determining whether the T cells of said T cell population recognise the peptide(s) and/or analogue(s). The method may performed in vivo. Peptides and a kit which enable the method to be carried out are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/489,621, filed Jun. 6, 2012, allowed, which is acontinuation application of U.S. patent application Ser. No. 13/013,466,filed Jan. 25, 2011, now U.S. Pat. No. 8,216,795, which is acontinuation application of U.S. patent application Ser. No. 12/579,019,filed Oct. 14, 2009, now U.S. Pat. No. 7,901,898, which is acontinuation application of U.S. patent application Ser. No. 09/830,839,filed Feb. 19, 2002, now U.S. Pat. No. 7,632,646, which is a U.S.National Phase Application under 35 U.S.C. §371 of International PatentApplication No. PCT/GB99/03635, filed Nov. 3, 1999, and published inEnglish on May 11, 2000, as International Publication No. WO00/26248,which claims priority benefit from Great Britain Application No.9824213.4, filed Nov. 4, 1998, and U.S. Provisional Patent ApplicationNo. 60/107,004, filed Nov. 4, 1998, the contents of all of which areincorporated by reference herein.

SEQUENCE LISTING

The specification further incorporates by reference a substituteSequence Listing submitted via EFS on Oct. 14, 2009. Pursuant to 37C.F.R. §1.52(e)(5), the substitute Sequence Listing text file,identified as 0775290121.txt, is 2,788 bytes and was created on Oct. 14,2009. The substitute Sequence Listing, electronically filed herewith,does not extend beyond the scope of the specification and thus does notcontain new matter.

BACKGROUND

The invention relates to a method of diagnosis of mycobacterialinfection, particularly Mycobacterium tuberculosis infection. It alsorelates to peptides and a kit which can be used to carry out thediagnostic method

Current diagnostic tests for tuberculosis disease are either slow orunreliable. Tests that rely on the identification of the mycobacteriumwhich causes tuberculosis are slow because culturing of themycobacterium can take up to 8 weeks. In some cases it proves impossibleto culture the bacteria. In addition the obtaining of samples to detectthe presence of the mycobacterium often requires invasive procedures

An alternative test is the tuberculin skin test (TST) or Mantoux testwhich is based on the detection of a delayed type hypersensitivity (DTH)response to an intradermal administration of a Purified ProteinDerivative of the mycobacterium. Although this test takes less time thantests which rely on identification of the mycobacterium, it is lessreliable because of the widespread use of BCG as a vaccine againsttuberculosis. BCG is closely related to M. tuberculosis and thereforeindividuals who have been vaccinated with BCG can react positively to aTST. In addition a large proportion of people with active tuberculosisare not detected by a TST because of cutaneous immune anergy. Thus TSThas a low specificity and sensitivity.

Using an assay which detects release of IFN-γ from T cells the inventorshave found 8 peptides from the ESAT-6 protein of M. tuberculosis whichare recognised by the T cells of a high proportion of patients withtuberculosis, and in particular the peptide represented by SEQ ID NO: 1is recognised by 57% of patients tested and 68% of healthy contactstested. These contacts have been exposed to open pulmonary tuberculosis.The inventors have combined these peptides into a panel of peptideswhich when used together in a diagnostic test provide a specificity of91.5%, and a sensitivity of 96%. The inventors have also found threeother peptides from ESAT-6 which are recognised by the T cells ofpatients with tuberculosis which can be used to increase the sensitivityof the diagnostic test.

Advantageously BCG does not have the ESAT-6 gene and therefore unlikeprevious tests, including TST, the diagnostic test can distinguishbetween patients with tuberculosis and patients who have been vaccinatedwith BCG.

Brandt et al. (1996), Journal of Immunology, 157, 3527-33 disclosesepitopes from ESAT-6 which are recognised by mice. However it is notpossible to predict based on the epitopes which are recognised in micewhich epitopes will be recognised in humans. As well as otherdifferences in epitope processing, presentation and recognition micehave different MHC molecules from humans, and thus are expected torecognise different epitopes from humans. This is demonstrated by thefact that Brandt et al find the recognition of epitopes in mice whichare not found to be recognised in humans by the present inventors.

SUMMARY OF THE INVENTION

The invention provides a method of diagnosing infection in a host, orexposure of a host, to a mycobacterium which expresses ESAT-6 comprising(i) contacting a population of T cells from the host with one or morepeptides or analogues selected from the peptides represented by SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, and analogues thereof which canbind a T cell receptor which recognises any of the said peptides, butnot (a) SEQ ID NO: 3 or 5 or an analogue thereof alone, nor (b) acombination of peptides and/or analogues selected from SEQ ID NO: 3 and5 and analogues thereof; and (ii) determining whether the T cells ofsaid T cell population recognise the peptide(s) and/or analogue(s).Preferably at least the peptide represented by SEQ ID NO: 1 or ananalogue thereof is used. In other preferred embodiments at least all ofthe peptides represented: by SEQ ID NO: 1, 5, 6 and 8; or by SEQ ID NO's1 to 8 are used.

The invention also provides a kit for carrying out the method comprisingone or more of the peptides or analogues and optionally a means todetect the recognition of the peptide by the T cell.

The invention additionally provides a peptide with the sequence of SEQID NO: 1, 2, 4, 6, 7, 8, 9, 10 or 11, or an analogue thereof, and apolynucleotide which is capable of being expressed to provide thepeptide or analogue.

DETAILED DESCRIPTION OF THE INVENTION

The sequences of SEQ ID NOs 1 to 11 are shown below:

SEQ ID NO 1: MTEQQWNFAGIEAAA (ES 1) SEQ ID NO 2: SAIQGNVTSIHSLLD (ES4)SEQ ID NO 3: QKWDATATELNNALQ (ES12) SEQ ID NO 4: NNALQNLARTISEAG (ES14)SEQ ID NO 5: NLARTISEAGQAMAS (ES15) SEQ ID NO 6: WNFAGIEAAASAIQG (ES2)SEQ ID NO 7: EGKQSLTKLAAAWGG (ES7) SEQ ID NO 8: YQGVQQKWDATATEL (ES11)SEQ ID NO 9: NVTSIHSLLDEGKQS (ES5) SEQ ID NO 10: IEAAASAIQGNVTSI (ES3)SEQ ID NO 11: TATELNNALQNLART (ES13)

The host is generally a human but may be an animal, typically one whichcan be naturally or artificially infected by a mycobacterium. The hostmay be a mammal, such as a primate, cow, sheep, pig, badger or rodent,e.g. a mouse or rat. The host typically has an active or latentmycobacterial infection, or has had such an infection recently. The hostmay test positive or negative in a Mantoux test. The host may be at riskof a mycobacterial infection, typically for socio-economic reasons ormay have a genetic or acquired predisposition to mycobacterialinfection.

The host may be a healthy contact who has been exposed to amycobacterium. Typically the exposure is to pulmonary tuberculosis, suchas ‘open’ pulmonary tuberculosis which is sputum a. f. b. (acid-fastbacillus) smear positive. Thus the method may be used to trace thehealthy contacts of individuals with such tuberculosis infections. Themethod may also be used to carry out population surveys to measure thenumber of individuals in a population who have a mycobacterial infectionor are healthy contacts.

The mycobacterium expresses ESAT-6. Generally, the ESAT-6 has a sequencewhich comprises one or more of the sequences represented by SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or one or more homologues of thesesequences. Such homologues can bind a T cell receptor which recognisesthe equivalent peptide represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or 11 and/or can inhibit the binding to a T cell receptor of thesaid equivalent peptide.

The mycobacterium is generally M. tuberculosis. The mycobacterium may beM. marinum or M. kansasii. The pattern of clinical symptoms can be usedto distinguish between these two organisms and M. tuberculosis. Themycobacterium may be M. bovis. This is able to infect humans.

The T cells which recognise the peptide in the method are generally Tcells which have been pre-sensitised in vivo to antigen from amycobacterium. These antigen-experienced T cells are generally presentin the peripheral blood of a host which has been exposed to themycobacterium at a frequency of 1 in 10⁶ to 1 in 10³ peripheral bloodmononuclear cells (PBMCs). The T cells may be CD4 and/or CD8 T cells.

It is understood that the term ‘peptide’ as used herein also includesthe analogue of that peptide (which may not be a peptide as defined bythe ordinary use of the term) unless the context requires otherwise.

In the method the T cells can be contacted with the peptides in vitro orin vivo, and determining whether the T cells recognise the peptide canbe done in vitro or in vivo. Thus the invention provides a method ofdiagnosis which is practised on the human or animal body. The inventionalso provides one or more of the peptides or analogues selected from thepeptides represented by SEQ ID NO: 5, 6, 7, 8, 9, 10 or 11 and analoguesthereof which can bind a T cell receptor that recognises any of the saidpeptides, but not (a) SEQ ID NO: 3 or 5 or an analogue thereof alone,nor (b) a combination of peptides and/or analogues selected from SEQ IDNO: 3 and 5 and analogues thereof, for use in diagnosing in a hostinfection by or exposure to a mycobacterium which expresses ESAT-6, saidmethod comprising determining whether T cells of the host recognise thepeptide(s) and/or analogue(s).

Determination of whether the T cells recognise the peptide is generallydone by detecting a change in the state of the T cells in the presenceof the peptide or determining whether the T cells bind the peptide. Thechange in state is generally caused by antigen specific functionalactivity of the T cell after the T cell receptor binds the peptide.Generally when binding the T cell receptor the peptide is bound to anMHC class II molecule, which is typically present on the surface of anantigen presenting cell (APC).

The change in state of the T cell may be the start of or increase insecretion of a substance from the T cell, such as a cytokine, especiallyIFN-γ, IL-2 or TNF-α. Determination of IFN-γ secretion is particularlypreferred. The substance can typically be detected by allowing it tobind to a specific binding agent and then measuring the presence of thespecific binding agent/substance complex. The specific binding agent istypically an antibody, such as polyclonal or monoclonal antibodies.Antibodies to cytokines are commercially available, or can be made usingstandard techniques.

Typically the specific binding agent is immobilised on a solid support.After the substance is allowed to bind the solid support can optionallybe washed to remove material which is not specifically bound to theagent. The agent/substance complex may be detected by using a secondbinding agent which will bind the complex. Typically the second agentbinds the substance at a site which is different from the site whichbinds the first agent. The second agent is preferably an antibody and islabelled directly or indirectly by a detectable label.

Thus the second agent may be detected by a third agent which istypically labelled directly or indirectly by a detectable label. Forexample the second agent may comprise a biotin moiety, allowingdetection by a third agent which comprises a streptavidin moiety andtypically alkaline phosphatase as a detectable label.

In one embodiment the detection system which is used is the ex-vivoELISPOT assay described in WO 98/23960. In that assay IFN-γ secretedfrom the T cell is bound by a first IFN-γ specific antibody which isimmobilised on a solid support. The bound IFN-γ is then detected using asecond IFN-γ specific antibody which is labelled with a detectablelabel. Such a labelled antibody can be obtained from MABTECH (Stockholm,Sweden). Other detectable labels which can be used are discussed below.

The change in state of the T cell which can be measured may be theincrease in the uptake of substances by the T cell, such as the uptakeof thymidine. The change in state may be an increase in the size of theT cells, or proliferation of the T cells, or a change in cell surfacemarkers on the T cell.

Generally the T cells which are contacted in the method are taken fromthe host in a blood sample, although other types of samples whichcontain T cells can be used. The sample may be added directly to theassay or may be processed first. Typically the processing may comprisediluting of the sample, for example with water or buffer. Typically thesample is diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10fold.

The processing may comprise separation of components of the sample.Typically mononuclear cells (MCs) are separated from the samples. TheMCs will comprise the T cells and APCs. Thus in the method the APCspresent in the separated MCs can present the peptide to the T cells. Inanother embodiment only T cells, such as only CD4 or only CD8 T cells,can be purified from the sample. PBMCs, MCs and T cells can be separatedfrom the sample using techniques known in the art, such as thosedescribed in Lalvani et at (1997) J. Exp. Med. 186, p 859-865.

Preferably the T cells used in the assay are in the form of unprocessedor diluted samples, or are freshly isolated T cells (such as in the formof freshly isolated MCs or PBMCs) which are used directly ex vivo, i.e.they are not cultured before being used in the method. However the Tcells can be cultured before use, for example in the presence of one ormore of the peptides, and generally also exogenous growth promotingcytokines. During culturing the peptides are typically present on thesurface of APCs, such as the APC used in the method. Pre-culturing ofthe T cells may lead to an increase in the sensitivity of the method.Thus the T cells can be converted into cell lines, such as short termcell lines (for example as described in Ota et al (1990) Nature 346, p183-187).

The APC which is typically present in the method may from the same hostas the T cell or from a different host. The APC may be a naturallyoccurring APC or an artificial APC. The APC is a cell which is capableof presenting the peptide to a T cell. It is typically a B cell,dendritic cell or macrophage. It is typically separated from the samesample as the T cell and is typically co-purified with the T cell. Thusthe APC may be present in MCs or PBMCs. The APC is typically a freshlyisolated ex vivo cell or a cultured cell. It may be in the form of acell line, such as a short term or immortalised cell line. The APC mayexpress empty MHC class II molecules on its surface.

Typically in the method the T cells derived from the sample can beplaced into an assay with all the peptides (i.e. a pool of the peptides)which it is intended to test (the relevant panel) or the T cells can bedivided and placed into separate assays each of which contain one ormore of the peptides. Preferably in the in vitro or in vivo forms of themethod at least the peptide represented by SEQ ID NO: 1 or an analoguethereof is used. Typically one or more, or all, of the peptidesrepresented by SEQ ID NOs 2, 3, 4, 5 and 6, preferably also 7 and/or 8,and in one embodiment also 9 and/or 10 and/or 111 are also used in themethod, leading to the method having an increased sensitivity. Inanother embodiment only the peptides represented by SEQ ID NOs 1, 2, 3,4, 5, 6, 8 and 9 are used in the method.

The invention also provides the peptides such as two or more of any ofthe peptides mentioned herein (for example in any of the combinationsmentioned herein) for simultaneous separate or sequential use (e.g. forin vivo use).

In one embodiment peptide per se is added directly to an assaycomprising T cells and APCs. As discussed above the T cells and APCs insuch an assay could be in the form of MCs. When peptides which can berecognised by the T cell without the need for presentation by APCs areused then APCs are not required. Analogues which mimic the originalpeptide bound to a MHC molecule are an example of such a peptide.

In one embodiment the peptide is provided to the APC in the absence ofthe T cell. The APC is then provided to the T cell, typically afterbeing allowed to present the peptide on its surface. The peptide mayhave been taken up inside the APC and presented, or simply be taken uponto the surface without entering inside the APC.

The duration for which the peptide is contacted with the T cells willvary depending on the method used for determining recognition of thepeptide. Typically 10⁵ to 10⁷, preferably 5×10⁵ to 10⁶ PBMCs are addedto each assay. In the case where peptide is added directly to the assayits concentration is from 10⁻¹ to 10³ μg/ml, preferably 0.5 to 50 μg/mlor 1 to 10 μg/ml.

Typically the length of time for which the T cells are incubated withthe peptide is from 4 to 24 hours, preferably 6 to 16 hours. When usingex vivo PBMCs it has been found that 0.3×10⁶ PBMCs can be incubated in10 μg/ml of peptide for 12 hours at 37° C.

The determination of the recognition of the peptide by the T cells maybe done by measuring the binding of the peptide to the T cells.Typically T cells which bind the peptide can be sorted based on thisbinding, for example using a FACS machine. The presence of T cells whichrecognise the peptide will be deemed to occur if the frequency of cellssorted using the peptide is above a ‘control’ value. The frequency ofantigen-experienced T cells is generally 1 and 10⁶ to 1 in 10³, andtherefore whether or not the sorted cells are antigen-experienced Tcells can be determined.

The determination of the recognition of the peptide by the T cells maybe measured in vivo. Typically the peptide is administered to the hostand then a response which indicates recognition of the peptide may bemeasured. In one embodiment the peptide is administered intradermally,typically in a similar manner to the Mantoux test. The peptide may beadministered epidermally. The peptide is typically administered byneedle, such as by injection, but can be administered by other methodssuch as ballistics, for example the ballistics techniques which havebeen used to deliver nucleic acids. EP-A-0693119 describes techniqueswhich can typically be used to administer the peptide. Typically from0.001 to 1000 μg, for example from 0.01 to 100 μg or 0.1 to 10 μg ofpeptide is administered.

Alternatively an agent can be administered which is capable of providingthe peptides in vivo. Thus a polynucleotide capable of expressing thepeptide can be administered, typically in any of the ways describedabove for the administration of the peptide. The polynucleotidetypically has any of the characteristics of the polynucleotide providedby the invention which is discussed below. Peptide is expressed from thepolynucleotide in vivo and recognition of the peptide in vivo ismeasured. Typically from 0.001 to 1000 μg, for example from 0.01 to 100μg or 0.1 to 10 μg of polynucleotide is administered. Recognition of thepeptide in vivo is typically indicated by the occurrence of a DTHresponse. This is generally measured by visual examination of the siteof administration of the peptide to determine the presence ofinflammation, such as by the presence of induration, erythema or oedema.

The analogue which can be used in the method can bind to a T cellreceptor which recognises the equivalent peptide represented by SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. Therefore generally when theanalogue is added to T cells in the presence of the equivalent saidpeptide, typically also in the presence of an APC, the analogue inhibitsthe recognition of the equivalent peptide. The binding of the analogueto the said T cell receptors can be tested by standard techniques. Forexample T cell receptors can be isolated from T cells which have beenshown to recognise the peptide (e.g. using the method of the invention).Demonstration of the binding of the analogue to the T cell receptors canthen shown by determining whether the T cell receptors inhibit thebinding of the analogue to a substance that binds the analogue, e.g. anantibody to the analogue. Typically the analogue is bound in an MHCmolecule in such an inhibition of binding assay.

Typically the analogue inhibits the binding of the peptide to a T cellreceptor. In this case the amount of peptide which can bind the T cellreceptor in the presence of the analogue is decreased. This is becausethe analogue is able to bind the T cell receptor and therefore competeswith the peptide for binding to the T cell receptor.

T cells for use in the above binding experiments can be isolated frompatients with mycobacterial infection, for example with the aid of themethod of the invention. Since whole ESAT-6 is unable to bind the T cellreceptor which recognises the peptide it is not encompassed by the term‘analogue’.

Other binding characteristics of the analogue are also the same as thepeptide, and thus typically the analogue binds to the same MHC class IImolecule which the peptide binds. The analogue of the peptiderepresented by SEQ ID NO: 1 typically binds HLA-DR1 and/or HLA-DR7. Theanalogue typically binds to antibodies specific for the peptide, andthus inhibits binding of the peptide to such an antibody.

The analogue is typically a peptide. It may have homology with theequivalent original peptide represented by one of SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11. A peptide which is homologous to anotherpeptide is typically at least 70% homologous to the peptide, preferablyat least 80 or 90% and more preferably at least 95%, 97% or 99%homologous thereto, for example over a region of at least 15, preferablyat least 30, for instance at least 40, 60 or 100 or more contiguousamino acids. Methods of measuring protein homology are well known in theart and it will be understood by those of skill in the art that in thepresent context, homology is calculated on the basis of amino acididentity (sometimes referred to as “hard homology”). For example theUWGCG Package provides the BESTFIT program which can be used tocalculate homology (for example used on its default settings) (Devereuxet al (1984) Nucleic Acids Research 12, p 387-395).

The homologous peptides typically differ by substitution, insertion ordeletion, for example from 1, 2, 3, 4, 5, 6, 7, 8 or more substitutions,deletions or insertions, which can be at the N or C terminal or at anyother position in the sequence. The substitutions are preferably‘conservative’. These are defined according to the following Table.Amino acids in the same block in the second column and preferably in thesame line in the third column may be substituted for each other:

ALIPHATIC Non-polar GAP ILV Polar-uncharged C S T M N Q Polar-charged DE KR AROMATIC H F W Y

The analogue is typically from 8 to 80 amino acids in length, such as 10to 60 or 12 to 50, preferably 15 to 30 or 20 to 25. Typically the aminoacids in the analogue at the equivalent positions to amino acids in theoriginal peptide which contribute to binding the MHC molecule or areresponsible for the recognition by the T cell receptor, are the same orare conserved.

Typically the analogue peptide comprises one or more modifications,which may be natural post-translation modifications or artificialmodifications. The modification may provide a chemical moiety (typicallyby substitution of a hydrogen, e.g. of a C—H bond), such as an amino,acetyl, hydroxy or halogen (e.g. fluorine) group or carbohydrate group.Typically the modification is present on the N or C terminus.

The analogue may comprise one or more non-natural amino acids, forexample amino acids with a side chain different from natural aminoacids. Generally, the non-natural amino acid will have an N terminusand/or a C terminus. The non-natural amino acid may be an L-amino acid.

The analogue typically has a shape, size, flexibility or electronicconfiguration which is substantially similar to the original peptide. Itis typically a derivative of the original peptide.

In one embodiment the analogue is or mimics the original peptide boundto a MHC class II molecule. The analogue may be or may mimic theoriginal peptide bound to 2, 3, 4 or more MHC class II moleculesassociated or bound to each other. These MHC molecules may be boundtogether using a biotin/streptavidin based system, in which typically 2,3 or 4 biotin labelled MHC molecules bind to a streptavidin moiety. Thisanalogue typically inhibits the binding of the peptides. Class IIcomplex to a T cell receptor or antibody which is specific for thecomplex. The analogue is typically an antibody or a fragment of anantibody, such as a Fab or (Fab).sub.2 fragment.

The analogue may be immobilised on a solid support, particularly ananalogue which mimics peptide bound to a MHC molecule.

The analogue is typically designed by computational means and thensynthesised using methods known in the art. Alternatively the analoguecan be selected from a library of compound. The library may be acombinatorial library or a display library, such as a phage displaylibrary. The library of compounds may be expressed in the displaylibrary in the form of being bound to a MHC class II molecule, such asthe MHC molecule which the original peptide binds. Analogues aregenerally selected from the library based on their ability to mimic thebinding characteristics of the original peptides. Thus they may beselected based on ability to bind a T cell receptor or antibody whichrecognises the original peptide.

The invention also provides a kit for carrying out the method comprisingone or more of the peptides or analogues and optionally a means todetect the recognition of the peptide by the T cell. Typically thepeptides are provided for simultaneous, separate or sequential use.Typically the means to detect recognition allows or aids detection basedon the techniques discussed above.

Thus the means may allow detection of a substance secreted by the Tcells after recognition. The kit may thus additionally include aspecific binding agent for the substance, such as an antibody. The agentis typically specific for IFN-γ. The agent is typically immobilised on asolid support. This means that after binding the agent the substancewill remain in the vicinity of the T cell which secreted it. Thus‘spots’ of substance/agent complex are formed on the support, each spotrepresenting a T cell which is secreting the substance. Quantifying thespots, and typically comparing against a control, allows determinationof recognition of the peptide.

The kit also comprise a means to detect the substance/agent complex. Adetectable change may occur in the agent itself after binding thesubstance, such as a colour change. Alternatively a second agentdirectly or indirectly labelled for detection may be allowed to bind thesubstance/agent complex to allow the determination of the spots. Asdiscussed above the second agent may be specific for the substance, butbinds a different site on the substance than the first agent.

The immobilised support may be a plate with wells, such as a microtitreplate. Each assay can therefore be carried out in a separate well in theplate.

The kit may additionally comprise medium for the T cells, detectionagents or washing buffers to be used in the detection steps. The kit mayadditionally comprise reagents suitable for the separation from thesample, such as the separation of PBMCs or T cells from the sample. Thekit may be designed to allow detection of the T cells directly in thesample without requiring any separation of the components of the sample.

The kit may comprise an instrument which allows administration of thepeptide, such as intradermal or epidermal administration. Typically suchan instrument comprises one or more needles. The instrument may allowballistic delivery of the peptide. The peptide in the kit may be in theform of a pharmaceutical composition.

The kit may also comprise controls, such as positive or negativeControls. The positive control may allow the detection system to betested. Thus the positive control typically mimics recognition of thepeptide in any of the above methods. Typically in the kits designed todetermine recognition in vitro the positive control is a cytokine. Inthe kit designed to detect in vivo recognition of the peptide thepositive control may be antigen to which most individuals shouldresponse.

The kit may also comprise a means to take a sample containing T cellsfrom the host, such as a blood sample. The kit may comprise a means toseparate mononuclear cells or T cells from a sample from the host.

The invention also provides a peptide with the sequence of SEQ ID NO: 1,2, 4, 6, 7, 8, 9, 10 or 11 or an analogue thereof. The inventionprovides a diagnostic product or panel comprising one or more of thepeptides typically in the combinations discussed above. The product istypically a composition such as a pharmaceutical composition.

The invention also provides a polynucleotide which is capable ofexpression to provide a peptide with the sequence of SEQ ID NO: 1, 2, 4,6, 7, 8, 9, 10 or 11 or an analogue thereof. Typically thepolynucleotide is DNA or RNA, and is single or double stranded. Thepolynucleotide therefore comprises sequence which encodes the sequenceof SEQ ID NO: 1, 2, 4, 6, 7, 8, 9, 10 or 11. To the 5′ and 3′ of thiscoding sequence the polynucleotide of the invention has sequence orcodons which are different from the sequence or codons 5′ and 3′ tothese sequences in the ESAT-6 gene. Therefore the polynucleotide of theinvention does not comprise the sequence coding for the whole of ESAT-6or fragments of ESAT-6, other than sequence coding for fragmentsrepresented by SEQ ID NO: 1, 2, 4, 6, 7, 8, 9, 10 or 11.

5′ and/or 3′ to the sequence encoding the peptide the polynucleotide hascoding or non-coding sequence. Sequence 5′ and/or 3′ to the codingsequence may comprise sequences which aid expression, such astranscription and/or translation, of the sequence encoding the peptide.The polynucleotide may be capable of expressing the peptide in aprokaryotic or eukaryotic cell. In one embodiment the polynucleotide iscapable of expressing the peptide in a mammalian cell, such as a human,primate or rodent cell.

The polynucleotide may be incorporated into a replicable vector. Such avector is able to replicate in a suitable cell. The vector may be anexpression vector. In such a vector the polynucleotide of the inventionis operably linked to a control sequence which is capable of providingfor the expression of the polynucleotide. The vector may contain aselectable marker, such as the ampicillin resistance gene.

The polynucleotide, peptide or antibody (see below) of the invention, orthe agents used in the method (for example in the detection ofsubstances secreted from T cells) may carry a detectable label.Detectable labels which allow detection of the secreted substance byvisual inspection, optionally with the aid of an optical magnifyingmeans, are preferred. Such a system is typically based on an enzymelabel which causes colour change in a substrate, for example alkalinephosphatase causing a colour change in a substrate. Such substrates arecommercially available, e.g. from BIORAD. Other suitable labels includeother enzymes such as peroxidase, or protein labels, such as biotin; orradioisotopes, such as ³²P or ³⁵S. The above labels may be detectedusing known techniques.

Polynucleotides, peptides or antibodies (see below) of the invention maybe in substantially purified form. They may be in substantially isolatedform, in which case they will generally comprise (for example about orat least) 90%, such as (for example about or at least) 95, 97 or 99% ofthe polynucleotide, peptide or antibody in the preparation. Thesubstantially isolated peptides which are not peptides (as defined inthe normal sense of the word) generally comprise (for example about orat least) 90%, such as (for example about or at least) 95, 97 or 99% ofthe dry mass of the preparation. The polynucleotide or peptide aretypically substantially free of other cellular components orsubstantially free of other mycobacterial cellular components. Thepolynucleotide or peptide may be used in such a substantially isolated,purified or free form in the method or be present in such forms in thekit.

The peptide or any combination of the peptides (for example as mentionedhere) is provided for use in a method of diagnosis practised on thehuman or animal body. The combinations of peptides are provided forsimultaneous, separate or sequential use in such a method.

The peptide or polynucleotide may be in the form of a pharmaceuticalcomposition which comprises the peptide or polynucleotide and apharmaceutically acceptable carrier or diluent. Suitable carriers anddiluents include isotonic saline solutions, for examplephosphate-buffered saline. Typically the composition is formulated forintradermal or epidermal administration or for application by ballistictechniques. Thus the peptide or polynucleotide may be associated with acarrier particle for ballistic delivery.

The peptide of the invention can be made using standard syntheticchemistry techniques, such as by use of an automated synthesizer.

The peptide is typically made from a longer polypeptide e.g. a fusionprotein, which polypeptide typically comprises the sequence of thepeptide. The peptide may be derived from the polypeptide by for examplehydrolysing the polypeptide, such as using a protease; or by physicallybreaking the polypeptide. The polypeptide is typically ESAT-6, which mayhave been expressed recombinantly.

The peptide can also be made in a process comprising expression of apolynucleotide, such as by expression of the polynucleotide of theinvention. The expressed polypeptide may be further processed to producethe peptide of the invention. Thus the peptide may be made in a processcomprising cultivating a cell transformed or transfected with anexpression vector as described above under conditions to provide forexpression of the peptide or a polypeptide from which the peptide can bemade. The polynucleotide of the invention can be made using standardtechniques, such as by using a synthesiser.

The invention also provides use of a peptide or analogue of theinvention to produce an antibody specific to the peptide. This antibodyor any of the antibodies mentioned herein may be produced by raisingantibody in a host animal. Such antibodies will be specific to thepeptide or to the substances mentioned above which bind antibodies. Thepeptide or substances are referred to as the ‘immunogen’ below. Methodsof producing monoclonal and polyclonal antibodies are well-known. Amethod for producing a polyclonal antibody comprises immunising asuitable host animal, for example an experimental animal, with theimmunogen and isolating immunoglobulins from the serum. The animal maytherefore be inoculated with the immunogen, blood subsequently removedfrom the animal and the IgG fraction purified. A method for producing amonoclonal antibody comprises immortalising cells which produce thedesired antibody. Hybridoma cells may be produced by fusing spleen cellsfrom an inoculated experimental animal with tumour cells (Kohler andMilstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by aconventional procedure. The hybridomas may be grown in culture orinjected intraperitoneally for formation of ascites fluid or into theblood stream of an allogenic host or immunocompromised host. Humanantibody may be prepared by in vitro immunisation of human lymphocytes,followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, theexperimental animal is suitably a goat, rabbit, rat or mouse. Ifdesired, the immunogen may be administered as a conjugate in which theimmunogen is coupled, for example via a side chain of one of the aminoacid residues, to a suitable carrier. The carrier molecule is typicallya physiologically acceptable carrier. The antibody obtained may beisolated and, if desired, purified.

The invention is illustrated by the following Examples:

Example 1 The Subjects Studied

Patients and controls were recruited prospectively at Northwick Park andSt Mark's NHS Trust, London, and the hospitals of the Oxford RadcliffeNHS Trust, Oxford, over a 16 month period from October 1997. A singleheparinised blood sample was drawn from each subject.

Patients with compatible clinical and radiographic findings who werebacteriologically confirmed with positive cultures for M. tuberculosisfrom one or more clinical specimens were recruited as tuberculosiscases. 29/47 patients (62%) were either untreated at the time ofvenesection or had received less than one month's therapy; the remainderwere at later time points in their treatment course.

Control patients were group-matched for ethnicity, age (within 4 years)and sex, and comprised individuals with a wide range of infectious,inflammatory, granulomatous, autoimmune and neoplastic conditions (table4). These included diseases that can be clinically difficult todifferentiate from tuberculosis and others which, like tuberculosis, canpresent as pyrexia of unknown origin. Patients with a past history oftuberculosis and those who reported recent contact with a known case oftuberculosis were excluded. None of the tuberculosis or control patientshad any clinical features to suggest HIV infection.

Example 2 Results Using the ELISPOT Assay

The ELISPOT assay was used to detect ex vivo antigen-experienced CD4 Tcells specific for ESAT-6. 17 peptides spanning the length of the ESAT-6molecule were synthesised by solid-phase f-moc chemistry (RESEARCHGENETICS, Alabama, USA and ZINSSER ANALYTICAL Frankfurt, Germany). Eachpeptide was 15 amino acids in length and overlapped its adjacent peptideby 10 residues. Identity was confirmed by mass spectrometry and purityby high performance liquid chromatography. Eight peptides werefrequently recognised epitopes; every subject who responded to any ofthe 17 ESAT-6-derived peptides also responded to at least one of thepanel of 8 peptides represented by SEQ ID NOs 1 to 8. Sequence homologysearches of the SWISSPROT and translated GENBANK databases of all knownprotein sequences confirmed that the sequences of these peptides areuniquely restricted to the ESAT-6 protein of M. tuberculosis complex. Aresponse to one or more of the peptides in this panel, testedindividually, was scored as indicative of M. tuberculosis infection.

Patients were also found to respond to the peptides represented by SEQID NOs 9 to 11.

Ex Vivo ELISPOT Assay for Single Cell IFN-γ Release: Enumeration ofCirculating ESAT-6 Peptide-Specific T Cells from Peripheral Blood

Based on the principle of a sandwich capture ELISA, the ELISPOT assaycaptures and detects IFN-γ molecules in the immediate vicinity of the Tcell from which they are secreted, while still at a relatively highconcentration. Following development, each resulting spot thus representthe “footprint” of an individual antigen-specific IFN-γ-secreting Tcell, or spot-forming cell (SFC). The ex vivo ELISPOT assay for IFN issufficiently sensitive to detect antigen-specific T cells directly fromperipheral blood without the need for a prior in vitro stimulation step(Lalvani et al (1997) J. Exp. Med. 186 p 859-865). Moreover, since theex vivo ELISPOT assay enumerates antigen-specific T cells with rapideffector function, only short incubation periods are required

Peripheral blood mononuclear cells (PBMC) were separated from 12 mlsblood by standard means as described in Lalvani et al (see above) andsuspended in RPMI supplemented with L-glutamine 2 mM, penicillin 100μg/ml and 10% heat-inactivated foetal calf serum (SIGMA, St. Louis, Mo.,USA) (R10).

Ninety-six-well polyvinylidene difluoride (PVDF)-backed plates(MILLIPORE) precoated with the anti-IFN-γ mAB 1-D1K at 15 μg/ml(MABTECH, Stockholm) were washed with RPMI medium 1640 and blocked withR10 for 1 3×10⁵ PBMC were added in 100 μl R10/well to the pre-coatedplates and peptides were added individually to each well at a finalconcentration 10 μg/ml. PPD (Batch RT49, STAATENS SERUMINSTITUT,Copenhagen, Denmark) was also added at 20 μg/ml. Phytohaemagglutinin(ICN BIOMEDICALS, Aurora, Ohio, USA) at 5 μg/ml was added to positivecontrol wells and no peptide was added to the negative control wells.Additionally, whole recombinant ESAT-6 was added at 10 μg/ml for 17tuberculosis patients and patients and all controls.

Assays were incubated for 6-12 hrs at 37° C., 5% CO.sub.2 and arrestedby washing×6 with PBS 0.05% Tween-20 (SIGMA, St. Louis, Mo., USA). Next,100 μl of 1 μg/ml of the biotinylated anti-IFN-γ mAb 7-B6-1-biotin(MABTECH, Stockholm, Sweden) was added. After 2 hrs incubation at roomtemperature, plates were washed again×6 and a 1:1000 dilution ofstreptavidin-alkaline phosphate conjugate (MABTECH, Stockholm, Sweden)was added to the wells and the plates incubated for a further hour.Next, wells were again washed×6 and 100 μl of chromogenic alkalinephosphatase substrate (BIORAD, Hercules, Calif., USA), diluted 1:25 withdeionized water, was added. After 30 mins the calorimetric reaction wasterminated by washing with tap water and plates allowed to dry.

Responses were scored as positive only if the test well contained atleast 5 IFN-γ SFCs more than the negative control wells and additionallythis number was at least twice that in negative control wells. Thiscut-off point (5 IFN-γ SFCs per 3×10⁵ PBMC) translates into a lowerthreshold of detection of 17 peptide-specific T cells per million PBMC,or 1/59,000 PBMC. Although the person performing the assays was notblind to the tuberculosis status of the patients, the read-out in SFCsis quantitative, our criteria for a positive response were stringent andbackground numbers of SFCs in the negative control wells were alwaysbelow 3, so that positive responses were objective and clear-cut. In allcases, positive and negative responses were immediately recognisable bydirect inspection of the plate, prior to precise enumeration with amagnifying glass.

The accuracy of the ESAT-6 peptide-based test as applied to thediagnosis of active tuberculosis was calculated and expressed assensitivity, specificity (confidence intervals calculated from thestandard binomial) and as likelihood ratios. The latter were thenapplied to a typical clinical scenario where tuberculosis is considereda diagnostic possibility with a pre-test probability of 20%.

Pools of peptides were also used in the above assays and were found tobe as effective in detecting responses as the same peptides testedindividually in separate assays.

Example 3 Demographic and Clinical Features of Patients and Controls

The tuberculosis patients represent the broad ethnic mix characteristicof tuberculosis in the UK, with a high prevalence of disease amongstpersons from the Indian Subcontinent (ISC) and blacks (table 2). Thecontrol patients were closely matched for ethnic origin, age and sexratio (table 2) and their diagnoses are listed in table 4. Thetuberculosis patients are representative of the broad clinical spectrumof disease caused by M. tuberculosis and 22/47 had extrapulmonarytuberculosis (table 3). Of those with pulmonary tuberculosis, 6/25 weresputum smear negative for acid fact bacilli. Thus immediate presumptivediagnosis by sputum microscopy was not possible in 28/47 (60%) ofpatients.

Prompt Diagnosis of M. tuberculosis Infection by Detection ofESAT-6-Specific T Cells in Blood 45/47 (96%) tuberculosis patientsresponded to one or more of the panel of 8 peptides shown in table 1 inthe ex vivo ELISPOT assay for IFN-γ (table 5). An unusually highproportion of patients responded to ES1. The inventors have shown thatthis peptide binds HLA DR1 and DR7.

Frequencies of ESAT-6 peptide-specific IFN-γ-secreting T cells weregenerally high, with a median of 200 ESAT-6 peptide-specific T cells permillion PBMC (inter-quartile range 105-596). IFN-γ SFCs specific foreach of the 8 peptides in table 1 are mainly CD4 T cells because T celllines have been generated against each of these peptides in vitro andpeptide-specific responses were abrogated by specific immunomagneticdepletion of CD4 T cells. ESAT-6-specific CD8 T cells are also detectedby peptides in the panel (e.g. ES14) that contain CD8 epitopes.

Only 4/47 (8.5%) controls with non-tuberculous illnesses responded toone or more of the panel of 8 ESAT-6-derived peptides; frequencies ofpeptide-specific IFN-γ-secreting T cells in these 4 responders weresimilar to those seen in tuberculosis patients. In all remainingcontrols there was a complete lack of response to all peptides. Use ofthe expanded set of 17 peptides spanning the entire length of ESAT-6gave identical results to those observed with the panel of 8 broadlyimmunogenic epitopes.

The 2 non-responders were pulmonary tuberculosis patients with advanceddisease and both were tested prior to treatment. Their clinical detailsare reviewed in the discussion. Of the 4 controls who responded, 2 hadacute pneumonia, one had acute bronchitis and the fourth had cellulitis.All 4 patients also had a strong ex vivo response to PPD in the ELISPOTassay for IFN-γ, indicating that they were sensitised to tuberculin.

Comparison of ESAT-6-Specific T Cell Responses with Responses to PPD 26tuberculosis patients underwent tuberculin skin testing with intradermalinoculation of 1 TU of PPD (NHS supply). Cutaneous induration at 72 hrswas measured with a ruler, and induration of 5 mm or more in diameterwas taken as positive, as per convention. Of these patients, only 18(69%) had a positive result on TST. By comparison, one third more, 24/26(92%), were positive by ex vivo ELISPOT for IFNγ and, overall, 45/47(96%) had a positive response by ex vivo ELISPOT (p=0.002, Fisher'sexact test). Although the control patients did not undergo tuberculinskin testing, 26/47 (55%) had positive responses to PPD in the ex vivoELISPOT assay for IFN-γ, indicating prior in vivo sensitisation of theirCD4 T cells to antigens in PPD.

Clinical Utility of the ESAT-6 Peptide-Based ELISPOT Assay for IFN-γ

The operational characteristics of this assay in this study are shown intable 5. These likelihood ratios generate large changes from pre-test topost-test probability. For example, if applied to a hypothetical patientwhere tuberculosis is considered a diagnostic possibility with apre-test probability of 20%, a positive test result would confer apositive predictive value of 74% while a negative test result would givea negative predictive value of 1%.

DISCUSSION

We have developed a highly accurate blood test for the rapid detectionof M. tuberculosis infection. The success of this ex vivo assay is basedon the sensitive detection of antigen-specific T cells using a highlyimmunogenic antigen that is highly specific for M. tuberculosis. Whenapplied as a diagnostic test for bacteriologically confirmed activetuberculosis, this assay yields a sensitivity of 96% and a specificityof 91.5% in the patient population studies (table 5). The tuberculosispatients represent a broad ethnic mix, reflecting the epidemiology oftuberculosis in the UK and, among the ethnically matched controlpatients, there were many common diseases that can be difficult todistinguish from tuberculosis. The operational characteristics (table 5)of this assay are therefore likely to be generally applicable toclinical practice in the UK. The test requires a single venous bloodsample, is easy and quick to perform, needs no specialised laboratoryfacilities and generates results by the next day; it is thus potentiallywell suited to routine hospital laboratories and could be readilyautomated.

The TST and sputum microscopy for AFB are the only tests for immediatepresumptive diagnosis of tuberculosis in general use. The sensitivity ofthe TST among the 26 tuberculosis patients who were tested by thismethod was only 69%, significantly less than the 96% sensitivity of theESAT-6-based ex vivo ELISPOT (p=0.002). Given the multiple majorlimitations or the TST, the ex vivo ELISPOT assay appears to be asuperior means of rapidly detecting M. tuberculosis infection. In ourseries of patients, sputum microscopy would have detected only 40% ofcases, compared with 96% for the ex vivo ELISPOT, which also detectedall 6 cases of sputum smear negative pulmonary tuberculosis.Furthermore, sputum microcopy cannot differentiate between M.tuberculosis and atypical mycobacteria. Since the ESAT-6 gene isrestricted to M. tuberculosis complex, M. kansasii, M. marinum and M.szulgei, (of these only M. kansasii can cause disease clinically similarto tuberculosis) our ESAT-6-based test may prove to be more specificthan sputum microscopy.

A variety of blood tests aimed at diagnosing tuberculosis have beendeveloped in the past but none have proved sufficiently sensitive,specific and convenient to enter routine use. Serological assays havesuffered from the lack of a target antigen that is as species-specificas ESAT-6 and the sensitivity of these assays is generallydisappointing, especially in acute forms of tuberculosis (pulmonary,miliary and pleural). PCR of circulating M. tuberculosis complex DNA inPBMC has been investigated as a method for diagnosing tuberculosis. Forpulmonary tuberculosis, sensitivity ranges from 33%-95% for differentinvestigators; in the largest series, of 76 bacteriologically confirmedHIV negative patients, sensitivity was 41%-27%. For extrapulmonarytuberculosis, blood based PCR had a sensitivity of only 4-27%.

2/47 tuberculosis patients did not respond in the ex vivo ELISPOT assay.Both were pulmonary tuberculosis patients with advanced disease and hadbeen severely symptomatic for several months prior to diagnosis and bothwere cachectic. One, a 20 year old Asian man, had extensive sputum smearpositive cavitatory disease with pleural involvement and was anergic onTST; the other, a 22 year old African woman, was sputum smear negative,had a bronchopneumonic pattern radiographically and was positive on TST.Interestingly, both patients were lymphopaenic, but both wereHIV-negative and responded well to therapy. Chronic, advancedtuberculosis causes non-specific immunosupression which mightconceivably account for the lack of detectable ESAT-6-specificIFN-γ-secreting T cells in these patients, but for all four patientswith severe miliary disease, often associated with cutaneous anergy,nonetheless responded in the ex vivo ELISPOT assay. At present it isunclear why these two patients did not respond and they represent truefalse negatives.

Although 33/47 (70%) control patients with non-tuberculosis illnesseswere BCG-vaccinated (as indicated by the presence of a scar), only 4/47responded in the ex vivo ELISPOT assay and 3 of these were notBCG-vaccinated. This assay is thus the first to successfully distinguishbetween BCG-vaccinated and M. tuberculosis infected patients. None ofthe 4 control patients who responded had clinical or radiographicfeatures suggestive of tuberculosis; two had acute pneumonia, one hadacute bronchitis and one cellulitis; all responded to first-lineantibiotics. All 4 patients also had a strong ex vivo response to PPD inthe ELISPOT assay for IFN-γ, indicating that they were sensitised totuberculin. Importantly, all four were from countries of high endemicityfor tuberculosis; three were Asian immigrants to the UK from Kenya (andall return there regularly) and the fourth was a visitor from Ethiopia.Thus all four had significant risk factors for exposure to M.tuberculosis. In contrast, none of the 22 control patients who were bornin the UK gave a positive response. It likely that the 4 responderswere, in fact, infected with M. tuberculosis but clearly do not haveactive disease. Thus, although these responders represent falsepositives if the assay is applied as a diagnostic test of activetuberculosis, we believe that they are, in biological terms truepositives, since the test has correctly detected M. tuberculosisinfection. Unfortunately, this conclusion cannot be formally provensince there exists no definitive means of confirming subclinical M.tuberculosis infection in asymptomatic exposed contacts. In contrast tothe ESAT-6 peptides, ex vivo ELISPOT responses to PPD were common(26/47) throughout the control group, indicating prior in vivosensitisation of their CD4 T cells to antigens in PPD, probably as aresult of BCG vaccination or exposure to environmental mycobacteria.

Further studies will be needed to answer whether the ESAT-6 based exvivo ELISPOT maintains its high sensitivity and specificity in othersettings. In indicating prior in vivo sensitisation of their CD4 T cellsto antigens in PPD, probably are latently infected with Ad tuberculosis,the specificity of an assay based on the detection of an M.tuberculosis-sensitised cellular immune system might be lower than inthis study. However, the high sensitivity of the ex vivo ELISPOT meansthat it could still be used to rule out a diagnosis of tuberculosis. Theassay will also need to be validated in children, where tuberculosis isusually a primary infection and presents acutely; animal studies ofESAT-6-specific cellular immune responses indicate that ESAT-6 isespecially strongly recognised in the early phase of primary infection,suggesting that our assay should prove as effective in children as it isin adults. Finally, the ex vivo ELISPOT needs to be evaluated in aseparate study of HIV-infected tuberculosis patients.

Example 4 Detection of CD4 T Cell Responses in Healthy Contacts

The panel consisting of the peptides shown by SEQ ID Nos: 1 to 8 wasused to detect responses in 26 healthy household contacts of index caseswith sputum smear positive pulmonary tuberculosis. Responses weredetected in 22 of the contacts indicating that the panel was sensitiveenough to detect asymptomatic M. tuberculosis infection.

26 healthy volunteers (22 of whom were BCG vaccinated) were also testedand none responded to any of the peptides. Thus the panel was able tosuccessfully distinguish between asymptomatic infected contacts andhealthy BCG vaccines.

Example 5 Detection of CD4 T Cell Responses in Healthy Contacts Using aDifferent Panel

A different panel than the one used in the previous Examples was used todetect responses in healthy contacts using the ELISPOT assay. The panelconsisted of the peptides shown by SEQ ID Nos: 1 to 6, 8 and 9.

All contacts have prolonged exposure to an index case with smearpositive open pulmonary tuberculosis and have a positive Heaf test ofgrade 3 or 4. One of the contacts works in the same room (and on thesame shift) as an index at a factory. One contact was on the samehospital ward as the index for several days. All other contacts werefrom the same household as the index.

20 out of 22 contacts tested positive with the panel. 15 of the contactstested positive with ES1.

Example 6 Sensitivity of the Panel in Comparison with the Use of WholeESAT-6

A number of patients with tuberculosis were found to only have CD8 Tcell responses specific for ESAT-6, and no CD4 T cell responses for thisantigen. Since in a diagnostic test whole ESAT-6 will only elicit aresponse from CD4 T cells and not from CD8 T cells such patients (whoonly have CD8 T cell responses) could not be detected using a diagnostictest based on whole ESAT-6. However peptides are able to elicit aresponse from both CD4 and CD8 T cells and therefore those patientscould detected using the peptides of the invention. Thus the use of suchpeptides leads to a higher sensitivity of diagnostic test.

TABLE 1 % of TB patients responding to individual Peptide peptide ES1 57ES2 40 ES4 23 ES7 15 ES11 34 ES12 25 ES14 28 ES15 34

TABLE 2 Demographic characteristics of tuberculosis patients andcontrols with non-tuberculosis illnesses Tuberculosis Controls withnon-TB Patients (%) Illnesses (%) Ethnicity: ISC 24 (51) 24 (48) Black14 (30) 14 (28) White 8 (17) 9 (19) Oriental 1 (2) 0 (0) Total: 47 47Sex (M/F) 30/17 27/20 Age: mean 35 (18-74) 39 (17-75) (range) ISC* =Indian Subcontinent

TABLE 3 Clinical features of tuberculosis patients (all confirmedculture positive for M. tuberculosis No. of patients (%) Pulmonary TB(PTB) 25 Sputum smear negative 6 (13) Sputum smear positive 19 (40)Positive TST/Total no. of PTB patients tested 9/14 Extrapulmonary TB(EPTB) 22 Lymphadenitis 6 (13) Muskuloskeletal 6 (13) Miliary 3 (6)Gastrointestinal 3 (6) Pleural 3 (6) Meningeal & miliary 1 (2) PositiveTST/Total no. of EPTB patients tested 9/12 Overall positive TST/all PTBand EPTB 18/26 (69%) patients tested

TABLE 4 Diagnosis of controls with non-TB illnesses Diagnosis No. ofpatients Pneumonia 6 Sarcoidosis 3 Infective Endocarditis 3 Lymphoma 2Lung cancer 2 Chronic osteomyelitis 2 Ulcerative colitis 2 Crohn'sdisease 2 Infective enterocolitis 2 Malaria (P. falciparum, P. vivax) 2Chronic Liver Disease 2 Cellulitis 2 Haemaglobinopathies (SS, HbH) 2Pulmonary A. lumbridoides infection 1 Acute pancreatitis 1 Dengue fever1 Bladder schistosomiasis 1 SLE 1 Acute bronchitis 1 Meningococcaemia 1Tonsillitis 1 Sickle cell criais 1 Gastric ulcer 1 DermatitisHerpetiformis 1 Venous thrombosis 1 Nephrotic syndrome 1 Congestivecardiac failure 1

TABLE 5 Projected clinical usefulness of ex vivo ELISPOT with ESAT-6peptides for the diagnosis of active tuberculosis, based on itsoperational characteristics in this study Response Rates TB Patients45/47 Controls with non-TB illness  4/47 Sensitivity (95% CI)   96%(92%-100%) Specificity (95% CI*) 91.5% (86%-97%) Likelihood Ratios (LR)Positive LR 11.25 Negative LR 0.05 CI* = Confidence Interval

What is claimed:
 1. A method of in vitro diagnosis of Mycobacteriumtuberculosis infection in a host, comprising (a) keeping a population ofT cells isolated from said host in contact with a peptide panelcomprising one or more epitopes contained within one or more peptidesselected from the group consisting of: SEQ ID NO: 1, peptide SEQ ID NO:2, peptide SEQ ID NO: 3, peptide SEQ ID NO: 4, peptide SEQ ID NO: 5,peptide SEQ ID NO: 6, peptide SEQ ID NO: 7, and peptide SEQ ID NO: 8,and (b) detecting a recognition response by the T cells to the peptidepanel.
 2. The method of claim 1, wherein the peptide panel furthercomprises one or more epitopes contained within one or more peptidesselected from the group consisting of: peptide SEQ ID NO: 9, peptide SEQID NO: 10 and peptide SEQ ID NO:
 11. 3. The method of claim 1, whereinthe recognition response is a change in the state of the T cells uponcontact with the peptide panel.
 4. The method of claim 1, wherein themethod yields a diagnostic sensitivity of at least 50%.
 5. The method ofclaim 1, wherein the T cells are incubated with the peptide panel forbetween 4 and 24 hours prior to determining the recognition response. 6.The method of claim 1, wherein the T cells are freshly isolated.
 7. Themethod of claim 1, wherein the T cells are isolated from blood.
 8. Themethod of claim 1, wherein the T cells comprise CD4+ and CD8+ T cells.9. The method of claim 1, wherein the T cells comprise CD4+ immediateeffector T cells.
 10. The method of claim 1, wherein the recognitionresponse is measured by detecting IFNγ production by the T cells. 11.The method of claim 1, wherein the recognition response is an increasein IFNγ production by the T cells.
 12. The method of claim 2, whereinthe method yields a diagnostic sensitivity of at least 50%.
 13. Themethod of claim 2, wherein the T cells are incubated with the peptidepanel for between 4 and 24 hours prior to determining the recognitionresponse.
 14. The method of claim 2, wherein the T cells are freshlyisolated.
 15. The method of claim 2, wherein the T cells are isolatedfrom blood.
 16. The method of claim 2, wherein the T cells comprise CD4+and CD8+ T cells.
 17. The method of claim 2, wherein the T cellscomprise CD4+ immediate effector T cells.
 18. The method of claim 2,wherein the recognition response is measured by detecting IFNγproduction by the T cells.
 19. A method of in vitro diagnosis ofMycobacterium tuberculosis infection in a host, comprising (a) keeping apopulation of T cells isolated from said host in contact with a peptidepanel comprising one or more epitopes contained within one or morepeptides selected from the group consisting of: SEQ ID NO: 1, peptideSEQ ID NO: 2, peptide SEQ ID NO: 3, peptide SEQ ID NO: 4, peptide SEQ IDNO: 5, peptide SEQ ID NO: 6, peptide SEQ ID NO: 7, and peptide SEQ IDNO: 8, peptide SEQ ID NO: 9, peptide SEQ ID NO: 10 and peptide SEQ IDNO: 11, and (b) detecting a recognition response by the T cells to thepeptide panel.
 20. The method of claim 19, wherein the recognitionresponse is a change in the state of the T cells upon contact with thepeptide panel.